Expression of plasminogen and microplasminogen in duckweed

ABSTRACT

The present invention provides methods and compositions for the production of recombinant plasminogen, microplasminogen, and fragments thereof in a duckweed expression system. It is the novel finding of the present invention that a duckweed expression system may be used to produce high levels of plasminogen and microplasminogen. The duckweed-produced plasminogen and microplasminogen can be activated to produce a polypeptide having protease activity. Thus, the invention encompasses methods for the expression of plasminogen, microplasminogen, and fragments thereof in duckweed, duckweed plants that are transformed with expression cassettes for the expression of plasminogen, microplasminogen, and fragments thereof, and nucleic acids comprising nucleotide sequences encoding plasminogen, microplasminogen, and fragments thereof, where these nucleotide sequences are modified to enhance their expression in duckweed.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/056,621, filed Feb. 11, 2005, which claims the benefit of U.S.Provisional Patent Application No. 60/543,487, filed Feb. 11, 2004, thecontents of each of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to recombinant protein production systems.More specifically, the invention is directed to methods and compositionsfor use in the expression of recombinant plasminogen and recombinantmicroplasminogen in duckweed.

BACKGROUND OF THE INVENTION

The duckweeds are the sole members of the monocotyledonous familyLemnaceae. The five genera and 38 species are all small, free-floating,fresh-water plants whose geographical range spans the entire globe(Landolt (1986) Biosystematic Investigation on the Family of Duckweeds:The Family of Lemnaceae—A Monograph Study Geobatanischen Institut ETH,Stiftung Rubel, Zurich). Although the most morphologically reducedplants known, most duckweed species have all the tissues and organs ofmuch larger plants, including roots, stems, flowers, seeds and fronds.Duckweed species have been studied extensively and a substantialliterature exists detailing their ecology, systematics, life-cycle,metabolism, disease and pest susceptibility, their reproductive biology,genetic structure, and cell biology. (Hillman (1961) Bot. Review 27:221; Landolt (1986) Biosystematic Investigation on the Family ofDuckweeds: The Family of Lemnaceae—A Monograph Study GeobatanischenInstitut ETH, Stiftung Rubel, Zurich).

The growth habit of the duckweeds is ideal for microbial culturingmethods. The plant rapidly proliferates through vegetative budding ofnew fronds, in a macroscopic manner analogous to asexual propagation inyeast. This proliferation occurs by vegetative budding from meristematiccells. The meristematic region is small and is found on the ventralsurface of the frond. Meristematic cells lie in two pockets, one on eachside of the frond midvein. The small midvein region is also the sitefrom which the root originates and the stem arises that connects eachfrond to its mother frond. The meristematic pocket is protected by atissue flap. Fronds bud alternately from these pockets. Doubling timesvary by species and are as short as 20-24 hours (Landolt (1957) Ber.Schweiz. Bot. Ges. 67: 271; Chang et al. (1977) Bull. Inst. Chem. Acad.Sin. 24:19; Datko and Mudd (1970) Plant Physiol. 65:16; Venkataraman etal. (1970) Z. Pflanzenphysiol. 62: 316).

Intensive culture of duckweed results in the highest rates of biomassaccumulation per unit time (Landolt and Kandeler (1987) The Family ofLemnaceae—A Monographic Study Vol. 2: Phytochemistry, Physiology,Application, Bibliography, Veroffentlichungen des GeobotanischenInstitutes ETH, Stiftung Rubel, Zurich), with dry weight accumulationranging from 6-15% of fresh weight (Tillberg et al. (1979) Physiol.Plant. 46:5; Landolt (1957) Ber. Schweiz. Bot. Ges. 67:271; Stomp,unpublished data). Protein content of a number of duckweed species grownunder varying conditions has been reported to range from 15-45% dryweight (Chang et al (1977) Bull. Inst. Chem. Acad. Sin. 24:19; Chang andChui (1978) Z. Pflanzenphysiol. 89:91; Porath et al. (1979) AquaticBotany 7:272; Appenroth et al. (1982) Biochem. Physiol. Pflanz.177:251). Using these values, the level of protein production per literof medium in duckweed is on the same order of magnitude as yeast geneexpression systems.

Duckweed plant or duckweed nodule cultures can be efficientlytransformed with an expression cassette containing a nucleotide sequenceof interest by any one of a number of methods includingAgrobacterium-mediated gene transfer, ballistic bombardment, orelectroporation. Stable duckweed transformants can be isolated bytransforming the duckweed cells with both the nucleotide sequence ofinterest and a gene that confers resistance to a selection agent,followed by culturing the transformed cells in a medium containing theselection agent. See U.S. Pat. No. 6,040,498 to Stomp et al.

A duckweed gene expression system provides the pivotal technology thatwould be useful for a number of research and commercial applications.For plant molecular biology research as a whole, a differentiated plantsystem that can be manipulated with the laboratory convenience of yeastprovides a very fast system in which to analyze the developmental andphysiological roles of isolated genes. For commercial production ofvaluable polypeptides, a duckweed-based system has a number ofadvantages over existing microbial or cell culture systems. Plantsdemonstrate post-translational processing that is similar to mammaliancells, overcoming one major problem associated with the microbial cellproduction of biologically active mammalian polypeptides, and it hasbeen shown by others (Hiatt (1990) Nature 334:469) that plant systemshave the ability to assemble multi-subunit proteins, an ability oftenlacking in microbial systems. Scale-up of duckweed biomass to levelsnecessary for commercial production of recombinant proteins is fasterand more cost efficient than similar scale-up of mammalian cells, andunlike other suggested plant production systems, e.g., soybeans andtobacco, duckweed can be grown in fully contained and controlled biomassproduction vessels, making the system's integration into existingprotein production industrial infrastructure far easier.

Accordingly, there remains a need for optimized methods and compositionsfor expressing proteins of interest in duckweed.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for theproduction of recombinant plasminogen, microplasminogen, and fragmentsthereof in a duckweed expression system. The duckweed expression systemof the present invention is optimized to produce high levels ofplasminogen, microplasminogen, and fragments thereof. Theduckweed-produced plasminogen and microplasminogen can be activated toproduce a polypeptide having protease activity. Thus, the inventionencompasses methods for the expression of plasminogen andmicroplasminogen in duckweed, duckweed plants that are transformed withexpression cassettes for the expression of plasminogen andmicroplasminogen, and nucleic acids comprising nucleotide sequencesencoding plasminogen and microplasminogen, where these nucleotidesequences are modified to enhance their expression in duckweed.

Accordingly, in one embodiment, the present invention provides a methodfor producing plasminogen in duckweed, wherein said method comprises thesteps of culturing a duckweed plant or duckweed plant cell, where theduckweed plant or duckweed plant cell is stably transformed with anucleic acid molecule comprising a nucleotide sequence encodingplasminogen; and collecting the plasminogen from said duckweed plant orduckweed plant cell. In some embodiments, the nucleotide sequenceencoding plasminogen is operably linked to a nucleotide sequenceencoding a signal peptide.

In another embodiment, the present invention provides a method forproducing microplasminogen in duckweed, where the method comprises thesteps of culturing within a duckweed culture medium a duckweed plantculture or a duckweed nodule culture, where the duckweed plant cultureor the duckweed nodule culture is stably transformed with a nucleic acidmolecule comprising a nucleotide sequence encoding microplasminogen andan operably linked coding sequence for a signal peptide that directssecretion of the microplasminogen into the culture medium; andcollecting the microplasminogen from the duckweed culture medium.

The invention also encompasses duckweed plants, duckweed nodules, andduckweed plant cells transformed with expression cassettes capable ofexpressing plasminogen or microplasminogen in duckweed. Also providedare nucleic acid molecules comprising a nucleotide sequence encodingplasminogen or microplasminogen, where the nucleotide sequences compriseduckweed-optimized codons.

These and other aspects of the present invention are disclosed in moredetail in the description of the invention given below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the level of plasminogen in tissue homogenates measured byELISA in 56 duckweed lines that were transformed with the plasminogenexpression construct BAP01. The level of plasminogen is expressed as apercentage of the total soluble protein in the homogenates. See Example1 in the Experimental section for additional details.

FIG. 2 shows the activity of plasmin produced by activatingduckweed-produced plasminogen with tPA. See Example 1 in theExperimental section for additional details.

FIG. 3 shows the quantitation of plasminogen by ELISA compared toplasmin activity after streptokinase activation. For the lines tested,quantitation by ELISA and streptokinase activity assay gave comparablevalues, indicating that Lemna-produced plasminogen has a similarspecific activity to the control protein. See Example 1 in theExperimental section for additional details.

FIG. 4 shows the stability of human plasminogen spiked into Lemna tissueextract. See Example 1 in the Experimental section for additionaldetails.

FIG. 5 shows the stability of Lemna plasminogen in tissue extract afterfreeze/thaw as measured by an activity assay. See Example 1 in theExperimental section for additional details.

FIG. 6 shows the formation of plasmin after urokinase or tPA activationof Lemna-produced plasminogen. “BAP” designates the Lemna-producedplasminogen. The left panel shows activation with tissue plasminogenactivator (tPA) and the right panel shows activation with urokinase(uK). See Example 1 in the Experimental section for additional details.

FIG. 7 shows the concentration of microplasminogen as measured by ELISAin media from 79 duckweed lines that were transformed with themicroplasminogen expression construct BAMP01. See Example 2 in theExperimental section for additional details.

FIG. 8 shows a zymogram analysis of Lemna-produced microplasminogenafter activation by tPA. The figure shows the presence of an activeproteolytic band that is not present in concentrated control media. SeeExample 2 in the Experimental section for additional details.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is drawn to methods and compositions for theproduction of recombinant plasminogen, microplasminogen, and fragmentsthereof in a duckweed expression system. It is the novel finding of thepresent invention that a duckweed expression system may be used toproduce high levels of plasminogen and microplasminogen. Theduckweed-produced plasminogen and microplasminogen can be activated toproduce a polypeptide having protease activity.

Plasminogen is the inactive precursor form of plasmin, the principalfibrinolytic enzyme in mammals. Plasmin also plays an important role incell migration, tissue remodeling, and bacterial invasion. Plasmin is aserine protease that preferentially cleaves Lys-|-Xaa and Arg-|-Xaabonds with higher selectivity than trypsin. Plasminogen activators suchas tissue plasminogen activator (tPA) or urokinase cleave humanplasminogen molecule at the Arg₅₆₀-Val₅₆₁ bond to produce activeplasmin. The two resulting chains of plasmin are held together by twointerchain disulphide bridges. The light chain (25 kDa) carries thecatalytic center (which comprises the catalytic triad) and sharessequence similarity with trypsin and other serine proteases. The heavychain (60 kDa) consists of five highly similar triple-loop structurescalled kringles. Some of the kringles contain lysine binding sites thatmediates the plasminogen/plasmin interaction with fibrin. Plasminbelongs to peptidase family S1.

Microplasminogen consists of the proenzyme domain of plasminogen with astretch of connecting peptide and a few residues of kringle 5 attachedat its N-terminal end. It is produced by the action of plasmin onplasminogen. See, for example, Shi et al. (1980) J. Biol. Chem.263:17071-5. Like plasminogen, microplasminogen is activated by tPA andurokinase to form a proteolytically active molecule. Human microplasminhas a molecular weight of approximately 29 kDa and has a lower affinityfor fibrin when compared with plasmin.

Plasmin and microplasmin are proposed for use in thrombolytic therapy ina number of applications including the treatment of myocardialinfarction, occlusive stroke, deep venous thrombosis, and peripheralarterial diseases. See, for example, U.S. Pat. No. 5,407,673, U.S. Pat.No. 6,355,243, U.S. Patent Application No. 20030175264, Lapchak et al.(2002) Stroke 33:2279-2284, and Nagai et al. (2003) J. Thromb. Haemost.1:307-13, each of which is herein incorporated by reference in itsentirety. One goal of using plasmin and microplasmin in such therapy isto avoid the side effects of therapy using plasminogen activators suchas tPA, urokinase, and streptokinase. Such side effects includegastrointestinal and intercranial hemorrhage. However, the use ofplasmin and microplasmin as therapeutic agents has been limited in partby the difficulty of producing large quantities of stable plasminogenand microplasminogen precursor proteins.

Although expression of large amounts of plasminogen in a recombinantsystem is a convenient way to obtain plasminogen for use in thrombolytictherapy, there have been great difficulties in expression of intacthuman plasminogen in expression systems due to the nearly ubiquitouspresence of intracellular plasminogen activators in mammalian celltypes. The presence of these activators results in the degradation ofthe produced plasminogen. See, for example, Busy et al. (1988)Fibrinolysis 2:64.

The present invention solves this problem by providing an expressionsystem capable of expressing high levels of stable plasminogen andmicroplasminogen. Thus, in one embodiment, the present inventionprovides a method for producing plasminogen in duckweed, wherein saidmethod comprises the steps of culturing a duckweed plant or duckweedplant cell, where the duckweed plant or duckweed plant cell is stablytransformed with a nucleic acid molecule comprising a nucleotidesequence encoding plasminogen; and collecting the plasminogen from saidduckweed plant or duckweed plant cell. The nucleotide sequence encodingplasminogen may be operably linked to a nucleotide sequence encoding asignal peptide.

The methods of the invention may be used to express high levels ofplasminogen in duckweed. Thus, in some embodiments of the method, atleast about 1%, at least about 2%, at least about 3%, at least about 4%,at least about 5%, at least about 6%, at least about 7%, or at leastabout 8% of the soluble protein in the duckweed plant or duckweed plantcell is plasminogen.

The present invention also provides an improvement in a method ofproducing stable plasminogen in duckweed, wherein the improvementcomprises producing the plasminogen in duckweed. The Lemna-producedplasminogen is stable and loses less than 10% of its activity whenstored overnight in a Lemna tissue extract. The Lemna-producedplasminogen also undergoes less than 10% degradation in Lemna tissueextracts following a freeze-thaw cycle.

In another embodiment, the present invention provides a method forproducing microplasminogen in duckweed, where the method comprises thesteps of culturing within a duckweed culture medium a duckweed plantculture or a duckweed nodule culture, where the duckweed plant cultureor the duckweed nodule culture is stably transformed with a nucleic acidmolecule comprising a nucleotide sequence encoding microplasminogen andan operably linked coding sequence for a signal peptide that directssecretion of the microplasminogen into the culture medium; andcollecting the microplasminogen from the duckweed culture medium.

The methods of the invention may be used to express high levels ofmicroplasminogen in duckweed. Thus, in some embodiments of the method,the, duckweed culture medium contains at least about 1 mg/L, at leastabout 2 mg/L, at least about 5 mg/L microplasminogen, at least about 10mg/L microplasminogen, at least about 15 mg/L microplasminogen, or atleast about 20 mg/L microplasminogen as determined by quantitativeWestern blotting.

The present invention provides a method for producing a plasminogenfragment in duckweed, wherein said method comprises the steps ofculturing a duckweed plant or duckweed plant cell, where the duckweedplant or duckweed plant cell is stably transformed with a nucleic acidmolecule comprising a nucleotide sequence encoding the plasminogenfragment; and collecting the plasminogen fragment from the duckweedplant, the duckweed plant cell, or the duckweed culture medium. Thenucleotide sequence encoding plasminogen may be operably linked to anucleotide sequence encoding a signal peptide.

In some embodiments of the methods of the present invention, the nucleicacid molecule comprising the nucleotide sequence encoding plasminogen,microplasminogen, or fragment thereof is modified to enhance itsexpression in duckweed. Examples of such modifications include the useof duckweed-preferred codons in the coding sequence for the plasminogenmicroplasminogen, the use of an operably linked nucleotide sequencecomprising a plant intron that is inserted upstream of the codingsequence; and the use of a leader sequence that increases thetranslation of the nucleotide sequence encoding plasminogen ormicroplasminogen. In some embodiments, two or more of thesemodifications are used in combination. Where the nucleotide sequenceencoding plasminogen is operably linked to a nucleotide sequenceencoding a signal peptide, the nucleotide sequence encoding the signalpeptide may also comprise duckweed-preferred codons.

The invention also encompasses duckweed plants, duckweed nodules, andduckweed plant cells transformed with expression cassettes capable ofexpressing plasminogen or microplasminogen in duckweed. Also providedare nucleic acid molecules comprising a nucleotide sequence encodingplasminogen or microplasminogen, where the nucleotide sequences compriseduckweed-optimized codons.

DEFINITIONS

“Polypeptide” refers to any monomeric or multimeric protein or peptide.

“Biologically active polypeptide” refers to a polypeptide that has thecapability of performing one or more biological functions or a set ofactivities normally attributed to the polypeptide in a biologicalcontext. Within the context of a protease precursor such as plasminogenor microplasminogen, biological activity encompasses the capability ofthe polypeptide to be activated to produce a proteolytically activemolecule. Proteolytic activity of plasminogen or microplasminogenfollowing activation may be measured by any assay known in the art,including the assay based on the use of a chromogenic substrate asdescribed elsewhere herein.

The terms “expression” or “production” refer to the biosynthesis of agene product, including the transcription, translation, and assembly ofsaid gene product.

The term “duckweed” refers to members of the family Lemnaceae. Thisfamily currently is divided into five genera and 38 species of duckweedas follows: genus Lemna (L. aequinoctialis, L. disperma, L.ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L.obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L.valdiviana); genus Spirodela (S. intermedia, S. polyrrhiza, S.punctata); genus Wolffia (Wa. angusta, Wa. arrhiza, Wa. australina, Wa.borealis, Wa. brasiliensis, Wa. columbiana, Wa. elongata, Wa. globosa,Wa. microscopica, Wa. neglecta); genus Wolfiella (Wl. caudata, Wl.denticulata, Wl. gladiata, Wl. hyalina, Wl. lingulata, Wl. repunda, Wl.rotunda, and Wl. neotropica) and genus Landoltia (L. punctata). Anyother genera or species of Lemnaceae, if they exist, are also aspects ofthe present invention. Lemna species can be classified using thetaxonomic scheme described by Landolt (1986) Biosystematic Investigationon the Family of Duckweeds: The family of Lemnaceae—A Monograph StudyGeobatanischen Institut ETH, Stiftung Rubel, Zurich.

The term “duckweed nodule culture” as used herein refers to a culturecomprising duckweed cells wherein at least about 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells are differentiatedcells. A “differentiated cell,” as used herein, is a cell with at leastone phenotypic characteristic (e.g., a distinctive cell morphology orthe expression of a marker nucleic acid or protein) that distinguishesit from undifferentiated cells or from cells found in other tissuetypes. The differentiated cells of the duckweed nodule culture describedherein form a tiled smooth surface of interconnected cells fused attheir adjacent cell walls, with nodules that have begun to organize intofrond primordium scattered throughout the tissue. The surface of thetissue of the nodule culture has epidermal cells connect to each othervia plasmadesmata.

“Duckweed-preferred codons” as used herein refers to codons that have afrequency of codon usage in duckweed of greater than 17%.

“Lemna-preferred codons” as used herein refers to codons that have afrequency of codon usage in the genus Lemna of greater than 17%.

“Lemna gibba-preferred codons” as used herein refers to codons that havea frequency of codon usage in Lemna gibba of greater than 17%.

“Translation initiation codon” refers to the codon that initiates thetranslation of the mRNA transcribed from the nucleotide sequence ofinterest.

“Translation initiation context nucleotide sequence” as used hereinrefers to the identity of the three nucleotides directly 5′ of thetranslation initiation codon.

“Secretion” as used herein refers to translocation of a polypeptideacross the plasma membrane of a host plant cell. In some embodiments ofthe present invention, the polypeptide is retained within the apoplast,the region between the plasma membrane and the cell wall. In otherembodiments, the polypeptide is translocated across cell wall of theplant host cell.

“Operably linked” as used herein in reference to nucleotide sequencesrefers to multiple nucleotide sequences that are placed in a functionalrelationship with each other. Generally, operably linked DNA sequencesare contiguous and, where necessary to join two protein coding regions,in reading frame.

A. Expression Cassettes

According to the present invention, stably transformed duckweed isobtained by transformation with an expression cassette comprising anucleotide sequence encoding plasminogen or a nucleotide sequenceencoding microplasminogen. The expression cassette comprises atranscriptional initiation region linked to the nucleic acid or gene ofinterest. Such an expression cassette is provided with a plurality ofrestriction sites for insertion of the nucleotide sequence encoding theprotein of interest to be under the transcriptional regulation of theregulatory regions. The expression cassette may encode a single gene ofinterest. In particular embodiments of the invention, the nucleic acidto be transferred contains two or more expression cassettes, each ofwhich encodes at least one gene of interest.

The transcriptional initiation region, (e.g., a promoter) may be nativeor homologous or foreign or heterologous to the host, or could be thenatural sequence or a synthetic sequence. By foreign, it is intendedthat the transcriptional initiation region is not found in the wild-typehost into which the transcriptional initiation region is introduced. Asused herein a chimeric gene comprises a coding sequence operably linkedto a transcription initiation region that is heterologous to the codingsequence.

Any suitable promoter known in the art can be employed according to thepresent invention (including bacterial, yeast, fungal, insect,mammalian, and plant promoters). For example, plant promoters, includingduckweed promoters, may be used. Exemplary promoters include, but arenot limited to, the Cauliflower Mosaic Virus ³⁵S promoter, the opinesynthetase promoters (e.g., nos, mas, ocs, etc.), the ubiquitinpromoter, the actin promoter, the ribulose bisphosphate (RubP)carboxylase small subunit promoter, and the alcohol dehydrogenasepromoter. The duckweed RubP carboxylase small subunit promoter is knownin the art (Silverthorne et al. (1990) Plant Mol. Biol. 15:49). Otherpromoters from viruses that infect plants, preferably duckweed, are alsosuitable including, but not limited to, promoters isolated from Dasheenmosaic virus, Chlorella virus (e.g., the Chlorella virus adeninemethyltransferase promoter; Mitra et al. (1994) Plant Mol. Biol. 26:85),tomato spotted wilt virus, tobacco rattle virus, tobacco necrosis virus,tobacco ring spot virus, tomato ring spot virus, cucumber mosaic virus,peanut stump virus, alfalfa mosaic virus, sugarcane baciliformbadnavirus and the like.

Finally, promoters can be chosen to give a desired level of regulation.For example, in some instances, it may be advantageous to use a promoterthat confers constitutive expression (e.g., the mannopine synthasepromoter from Agrobacterium tumefaciens). Alternatively, in othersituations, it may be advantageous to use promoters that are activatedin response to specific environmental stimuli (e.g., heat shock genepromoters, drought-inducible gene promoters, pathogen-inducible genepromoters, wound-inducible gene promoters, and light/dark-inducible genepromoters) or plant growth regulators (e.g., promoters from genesinduced by abscissic acid, auxins, cytokinins, and gibberellic acid) orother compounds such as ethanol or ethylene. As a further alternative,promoters can be chosen that give tissue-specific expression (e.g.,root, leaf, and floral-specific promoters).

The overall strength of a given promoter can be influenced by thecombination and spatial organization of cis-acting nucleotide sequencessuch as upstream activating sequences. For example, activatingnucleotide sequences derived from the Agrobacterium tumefaciens octopinesynthase gene can enhance transcription from the Agrobacteriumtumefaciens mannopine synthase promoter (see U.S. Pat. No. 5,955,646 toGelvin et al.). In the present invention, the expression cassette cancontain activating nucleotide sequences inserted upstream of thepromoter sequence to enhance the expression of the nucleotide sequenceof interest. In one embodiment, the expression cassette includes threeupstream activating sequences derived from the Agrobacterium tumefaciensoctopine synthase gene operably linked to a promoter derived from anAgrobacterium tumefaciens mannopine synthase gene (see U.S. Pat. No.5,955,646, herein incorporated by reference).

The transcriptional cassette includes in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, anucleotide sequence of interest, and a transcriptional and translationaltermination region functional in plants. Any suitable terminationsequence known in the art may be used in accordance with the presentinvention. The termination region may be native with the transcriptionalinitiation region, may be native with the nucleotide sequence ofinterest, or may be derived from another source. Convenient terminationregions are available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthetase and nopaline synthetase termination regions. Seealso Guerineau et al. (1991) Mol. Gen. Genet. 262:141; Proudfoot (1991)Cell 64:671; Sanfacon et al. (1991) Genes Dev. 5:141; Mogen et al.(1990) Plant Cell 2:1261; Munroe et al. (1990) Gene 91:151; Ballas etal. (1989) Nucleic Acids Res. 17:7891; and Joshi et al. (1987) NucleicAcids Res. 15:9627. Additional exemplary termination sequences are thepea RubP carboxylase small subunit termination sequence and theCauliflower Mosaic Virus 35S termination sequence and the ubiquitinterminator from many plant species. Other suitable termination sequenceswill be apparent to those skilled in the art.

The expression cassettes may contain more than one gene or nucleic acidsequence to be transferred and expressed in the transformed plant. Thus,each nucleic acid sequence will be operably linked to 5′ and 3′regulatory sequences. Alternatively, multiple expression cassettes maybe provided.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells or tissues. Selectablemarker genes include genes encoding antibiotic resistance, such as thoseencoding neomycin phosphotransferase II (NEO), neomycinphosphotransferase III and hygromycin phosphotransferase (HPT), as wellas genes conferring resistance to herbicidal compounds. Herbicideresistance genes generally code for a modified target proteininsensitive to the herbicide or for an enzyme that degrades ordetoxifies the herbicide in the plant before it can act. See DeBlock etal. (1987) EMBO J. 6:2513; DeBlock et al. (1989) Plant Physiol. 91:691;Fromm et al. (1990) BioTechnology 8:833; Gordon-Kamm et al. (1990) PlantCell 2:603; and Frisch et al. (1995) Plant Mol. Biol. 27:405-9. Forexample, resistance to glyphosphate or sulfonylurea herbicides has beenobtained using genes coding for the mutant target enzymes,5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetolactatesynthase (ALS). Resistance to glufosinate ammonium, boromoxynil, and2,4-dichlorophenoxyacetate (2,4-D) have been obtained by using bacterialgenes encoding phosphinothricin acetyltransferase, a nitrilase, or a2,4-dichlorophenoxyacetate monooxygenase, which detoxify the respectiveherbicides.

For purposes of the present invention, selectable marker genes include,but are not limited to, genes encoding neomycin phosphotransferase II(Fraley et al. (1986) CRC Critical Reviews in Plant Science 4:1),neomycin phosphotransferase III (Frisch et al. (1995) Plant Mol. Biol.27:405-9), cyanamide hydratase (Maier-Greiner et al. (1991) Proc. Natl.Acad. Sci. USA 88:4250); aspartate kinase; dihydrodipicolinate synthase(Perl et al. (1993) BioTechnology 11:715); bar gene (Toki et al. (1992)Plant Physiol. 100:1503; Meagher et al. (1996) Crop Sci. 36:1367);tryptophan decarboxylase (Goddijn et al. (1993) Plant Mol. Biol.22:907); neomycin phosphotransferase (NEO; Southern et al. (1982) J.Mol. Appl. Gen. 1:327); hygromycin phosphotransferase (HPT or HYG;Shimizu et al. (1986) Mol. Cell. Biol. 6:1074); dihydrofolate reductase(DHFR; Kwok et al. (1986) Proc. Natl. Acad. Sci. USA 83:4552);phosphinothricin acetyltransferase (DeBlock et al. (1987) EMBO J.6:2513); 2,2-dichloropropionic acid dehalogenase (Buchanan-Wollatron etal. (1989) J. Cell. Biochem. 13D:330); acetohydroxyacid synthase (U.S.Pat. No. 4,761,373 to Anderson et al.; Haughn et al. (1988) Mol. Gen.Genet. 221:266); 5-enolpyruvyl-shikimate-phosphate synthase (aroA; Comaiet al. (1985) Nature 317:741); haloarylnitrilase (WO 87/04181 to Stalkeret al.); acetyl-coenzyme A carboxylase (Parker et al. (1990) PlantPhysiol. 92:1220); dihydropteroate synthase (sulI; Guerineau et al.(1990) Plant Mol. Biol. 15:127); and 32 kDa photosystem II polypeptide(psbA; Hirschberg et al. (1983) Science 222:1346 (1983).

Also included are genes encoding resistance to: gentamycin (e.g., aacC1,Wohlleben et al. (1989) Mol. Gen. Genet. 217:202-208); chloramphenicol(Herrera-Estrella et al. (1983) EMBO J. 2:987); methotrexate(Herrera-Estrella et al. (1983) Nature 303:209; Meijer et al. (1991)Plant Mol. Biol. 16:807); hygromycin (Waldron et al. (1985) Plant Mol.Biol. 5:103; Zhijian et al. (1995) Plant Science 108:219; Meijer et al.(1991) Plant Mol. Bio. 16:807); streptomycin (Jones et al. (1987) Mol.Gen. Genet. 210:86); spectinomycin (Bretagne-Sagnard et al. (1996)Transgenic Res. 5:131); bleomycin (Hille et al. (1986) Plant Mol. Biol.7:171); sulfonamide (Guerineau et al. (1990) Plant Mol. Bio. 15:127);bromoxynil (Stalker et al. (1988) Science 242:419); 2,4-D (Streber etal. (1989) BioTechnology 7:811); phosphinothricin (DeBlock et al. (1987)EMBO J. 6:2513); spectinomycin (Bretagne-Sagnard and Chupeau, TransgenicResearch 5:131).

The bar gene confers herbicide resistance to glufosinate-typeherbicides, such as phosphinothricin (PPT) or bialaphos, and the like.As noted above, other selectable markers that could be used in thevector constructs include, but are not limited to, the pat gene, alsofor bialaphos and phosphinothricin resistance, the ALS gene forimidazolinone resistance, the HPH or HYG gene for hygromycin resistance,the EPSP synthase gene for glyphosate resistance, the Hml gene forresistance to the Hc-toxin, and other selective agents used routinelyand known to one of ordinary skill in the art. See Yarranton (1992)Curr. Opin. Biotech. 3:506; Chistopherson et al. (1992) Proc. Natl.Acad. Sci. USA 89:6314; Yao et al. (1992) Cell 71:63; Reznikoff (1992)Mol. Microbiol. 6:2419; Barkley et al. (1980) The Operon 177-220; Hu etal. (1987) Cell 48:555; Brown et al. (1987) Cell 49:603; Figge et al.(1988) Cell 52:713; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA86:5400; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549;Deuschle et al. (1990) Science 248:480; Labow et al. (1990) Mol. Cell.Biol. 10:3343; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072; Wyborskiet al. (1991) Nuc. Acids Res. 19:4647; Hillenand-Wissman (1989) Topicsin Mol. And Struc. Biol. 10:143; Degenkolb et al. (1991) Antimicrob.Agents Chemother. 35:1591; Kleinschnidt et al. (1988) Biochemistry27:1094; Gatz et al. (1992) Plant J. 2:397; Gossen et al. (1992) Proc.Natl. Acad. Sci. USA 89:5547; Oliva et al. (1992) Antimicrob. AgentsChemother. 36:913; Hlavka et al. (1985) Handbook of ExperimentalPharmacology 78; and Gill et al. (1988) Nature 334:721. Such disclosuresare herein incorporated by reference.

The above list of selectable marker genes are not meant to be limiting.Any lethal or non-lethal selectable marker gene can be used in thepresent invention.

B. Plasminogen and Microplasminogen

The present invention is directed to methods and compositions for theexpression of plasminogen, microplasminogen, and fragments thereof induckweed. The plasminogen to be expressed in duckweed may be from anymammalian source. In some embodiments, the plasminogen is human orporcine. In a particular embodiment, the plasminogen has the amino acidsequence of the human plasminogen shown in SEQ ID NO:4. In otherembodiments, the plasminogen is a biologically active variant of theamino acid sequence shown in SEQ ID NO:4.

Similarly, the microplasminogen to be expressed in duckweed may be fromany mammalian source. In some embodiments, the microplasminogen is humanor porcine. In a particular embodiment, the microplasminogen has theamino acid sequence of the human microplasminogen shown in SEQ ID NO:6.In other embodiments, the plasminogen is a biologically active variantof the amino acid sequence shown in SEQ ID NO6.

Fragments of plasminogen or microplasminogen may be produced accordingto the present invention. Such fragments may comprise at least 50, atleast 60, at least 70, at least 80, at least 90, at least 95, at least101, at least 150, at least 200, at least 250, at least 300, at least350, at least 377, at least 400, at least 450, at least 500, at least550, at least 600, at least 650, at least 700, or at least 750contiguous amino acids of a plasminogen protein. Examples of fragmentsthat may be produced according to the invention include miniplasminogenand angiostatin. Non-limiting examples of plasminogen ormicroplasminogen fragments are given in O'Reilly et al. (1994) Cell79:315-28; Sim et al. (1997) Cancer Res. 57:1329-34; U.S. Pat. No.5,972,896, and U.S. Patent Publications 20020164717, 20020037847, and20010016644; each of which is herein incorporated by reference in itsentirety. In some embodiments, the fragments retain the enzymaticactivity, for example the protease activity, of plasminogen ormicroplasminogen.

A “biologically active variant” of plasminogen or microplasminogen is apolypeptide derived from these polypeptides by deletion (so-calledtruncation) or addition of one or more amino acids to the N-terminaland/or C-terminal end of the native protein; deletion or addition of oneor more amino acids at one or more sites in the protein; or substitutionof one or more amino acids at one or more sites in the protein.Biologically active variant plasminogen and microplasminogenpolypeptides encompassed by the present invention are biologicallyactive, that is they are capable of being activated to produce a proteinhaving the protease activity of the plasmin family of proteases (EnzymeClass 3.4.21.7). Such biologically active variants may result from, forexample, genetic polymorphism or from human manipulation. Biologicallyactive variants of a plasminogen or microplasminogen according to theinvention will have at least about 50%, 60%, 65%, 70%, generally atleast about 75%, 80%, 85%, preferably at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, such as at least about 98%, 99% or more sequenceidentity to the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:6.Thus, a biologically active variant of plasminogen or microplasminogenof the invention may differ from the amino acid sequences shown in SEQID NO: 4 and SEQ ID NO:6 by as few as 1-15 amino acid residues, as fewas 1-10 amino acid residues, such as 6-10 amino acid residues, as few as5 amino acid residues, or as few as 4, 3, 2, or even 1 amino acidresidue. Examples of biologically active variants of plasminogen areknown in the art and are described, for example, in U.S. Pat. No.5,190,756. In order to retain biological activity, any substitutionswill preferably be conservative in nature, and truncations andsubstitutions will generally made in residues that are not required forprotease activity. The residues and domains underlying the activity ofplasmin/plasminogen and microplasmin/microplasminogen are known in theart and have been described, for example, in Kolev et al. (1997) J.Biol. Chem. 272:13666-675; de los Santos et al. (1997) Ciba Found. Symp.212:66-76, Peisach et al. (1999) Biochemistry 38:11180-11188, and Turneret al. (2002) J. Biol. Chem. 277:33-68-74); each of which is hereinincorporated by reference in its entirety.

The comparison of sequences and determination of percent identity andpercent similarity between two sequences can be accomplished using amathematical algorithm. In a preferred embodiment, the percent identitybetween two amino acid sequences is determined using the Needleman andWunsch (1970) J. Mol. Biol. 48:444-453 algorithm, which is incorporatedinto the GAP program in the GCG software package (available atwww.accelrys.com), using either a BLOSSUM62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentidentity between two nucleotide sequences is determined using the GAPprogram in the GCG software package, using a BLOSUM62 scoring matrix(see Henikoff et al. (1989) Proc. Natl. Acad. Sci. USA 89:10915) and agap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4,5, or 6. A particularly preferred set of parameters (and the one thatshould be used if the practitioner is uncertain about what parametersshould be applied to determine if a molecule is within a sequenceidentity limitation of the invention) is using a BLOSUM62 scoring matrixwith a gap weight of 60 and a length weight of 3).

The percent identity between two amino acid or nucleotide sequences canalso be determined using the algorithm of E. Meyers and W. Miller (1989)CABIOS 4:11-17 which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

C. Modification of Nucleotide Sequences for Enhanced Expression inDuckweed

In some embodiments, the present invention provides for the modificationof the expressed nucleotide sequence to enhance its expression induckweed. One such modification is the synthesis of the nucleotidesequence encoding plasminogen or microplasminogen usingduckweed-preferred codons. Methods are available in the art forsynthesizing nucleotide sequences with plant-preferred codons. See, forexample, U.S. Pat. Nos. 5,380,831 and 5,436,391; Perlak et al. (1991)Proc. Natl. Acad. Sci. USA 15:3324; Iannacome et al. (1997) Plant Mol.Biol. 34:485; and Murray et al., (1989) Nucleic Acids. Res. 17:477,herein incorporated by reference. The preferred codons may be determinedfrom the codons of highest frequency in the proteins expressed induckweed. Thus, the frequency of usage of particular a codon in duckweedmay be determined by analyzing codon usage in a group of duckweed codingsequences. A number of duckweed coding sequences are known to those ofskill in the art; see for example, the sequences contained in theGenBank® database which may be accessed through the website for theNational Center for Biotechnology Information, a division of theNational Library of Medicine, which is located in Bethesda, Md. Tablesshowing the frequency of codon usage based on the sequences contained inthe most recent GenBank® release may be found on the website for theKazusa DNA Research Institute in Chiba Japan; seewww.kazusa.or.jp/codon/. This database is described in Nakamura et al.(2000) Nucl. Acids Res. 28: 292.

It is recognized that genes that have been optimized for expression induckweed and other monocots can be used in the methods of the invention.See, e.g., EP 0 359 472, EP 0 385 962, WO 91/16432; Perlak et al. (1991)Proc. Natl. Acad. Sci. USA 88:3324; Iannacome et al. (1997) Plant Mol.Biol. 34:485; and Murray et al. (1989) Nuc. Acids Res. 17:477, and thelike, herein incorporated by reference. It is further recognized thatall or any part of the gene sequence may be optimized or synthetic. Inother words, fully optimized or partially optimized sequences may alsobe used. For example, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of thecodons may be duckweed-preferred codons. For example, in someembodiments, the nucleotide sequence encoding plasminogen ormicroplasminogen comprises between 50-100% duckweed-preferred codons orbetween 70-100% duckweed preferred codons. In one embodiment, between 90and 96% of the codons are duckweed-preferred codons. The coding sequenceof the nucleotide sequence of interest may comprise codons used with afrequency of at least 17% in duckweed. Codon usage in Lemna gibba(Table 1) and Lemna minor (Table 2) is shown below. In some embodiments,Table 1 or Table 2 is used to select duckweed preferred codons. Inparticular embodiments, the duckweed codon optimized sequence encodingplasminogen is the sequence shown in SEQ ID NO:3, and the duckweed codonoptimized sequence encoding microplasminogen is the nucleotide sequenceshown in SEQ ID NO:5.

TABLE 1 Lemna gibba codon usage from GenBank ® Release 139* Amino AcidCodon Number /1000 Fraction Gly GGG 57.00 28.89 0.35 Gly GGA 8.00 4.050.05 Gly GGT 3.00 1.52 0.02 Gly GGC 93.00 47.14 0.58 Glu GAG 123.0062.34 0.95 Glu GAA 6.00 3.04 0.05 Asp GAT 6.00 3.04 0.08 Asp GAC 72.0036.49 0.92 Val GTG 62.00 31.42 0.47 Val GTA 0.00 0.00 0.00 Val GTT 18.009.12 0.14 Val GTC 51.00 25.85 0.39 Ala GCG 44.00 22.30 0.21 Ala GCA14.00 7.10 0.07 Ala GCT 14.00 7.10 0.07 Ala GCC 139.00 70.45 0.66 ArgAGG 16.00 8.11 0.15 Arg AGA 11.00 5.58 0.10 Ser AGT 1.00 0.51 0.01 SerAGC 44.00 22.30 0.31 Lys AAG 116.00 58.79 1.00 Lys AAA 0.00 0.00 0.00Asn AAT 2.00 1.01 0.03 Asn AAC 70.00 35.48 0.97 Met ATG 67.00 33.96 1.00Ile ATA 4.00 2.03 0.06 Ile ATT 0.00 0.00 0.00 Ile ATC 63.00 31.93 0.94Thr ACG 19.00 9.63 0.25 Thr ACA 1.00 0.51 0.01 Thr ACT 6.00 3.04 0.08Thr ACC 50.00 25.34 0.66 Trp TGG 45.00 22.81 1.00 End TGA 4.00 2.03 0.36Cys TGT 0.00 0.00 0.00 Cys TGC 34.00 17.23 1.00 End TAG 0.00 0.00 0.00End TAA 7.00 3.55 0.64 Tyr TAT 4.00 2.03 0.05 Tyr TAC 76.00 38.52 0.95Leu TTG 5.00 2.53 0.04 Leu TTA 0.00 0.00 0.00 Phe TTT 4.00 2.03 0.04 PheTTC 92.00 46.63 0.96 Ser TCG 34.00 17.23 0.24 Ser TCA 2.00 1.01 0.01 SerTCT 1.00 0.51 0.01 Ser TCC 59.00 29.90 0.42 Arg CGG 23.00 11.66 0.22 ArgCGA 3.00 1.52 0.03 Arg CGT 2.00 1.01 0.02 Arg CGC 50.00 25.34 0.48 GlnCAG 59.00 29.90 0.86 Gln CAA 10.00 5.07 0.14 His CAT 5.00 2.53 0.26 HisCAC 14.00 7.10 0.74 Leu CTG 43.00 21.79 0.35 Leu CTA 2.00 1.01 0.02 LeuCTT 1.00 0.51 0.01 Leu CTC 71.00 35.99 0.58 Pro CCG 44.00 22.30 0.31 ProCCA 6.00 3.04 0.04 Pro CCT 13.00 6.59 0.09 Pro CCC 80.00 40.55 0.56

TABLE 2 Lemna minor codon usage from GenBank ® Release 139* AmAcid CodonNumber /1000 Fraction Gly GGG 8.00 17.39 0.22 Gly GGA 11.00 23.91 0.31Gly GGT 1.00 2.17 0.03 Gly GGC 16.00 34.78 0.44 Glu GAG 25.00 54.35 0.78Glu GAA 7.00 15.22 0.22 Asp GAT 8.00 17.39 0.33 Asp GAC 16.00 34.78 0.67Val GTG 21.00 45.65 0.53 Val GTA 3.00 6.52 0.07 Val GTT 6.00 13.04 0.15Val GTC 10.00 21.74 0.25 Ala GCG 13.00 28.26 0.32 Ala GCA 8.00 17.390.20 Ala GCT 6.00 13.04 0.15 Ala GCC 14.00 30.43 0.34 Arg AGG 9.00 19.570.24 Arg AGA 11.00 23.91 0.30 Ser AGT 2.00 4.35 0.05 Ser AGC 11.00 23.910.26 Lys AAG 13.00 28.26 0.68 Lys AAA 6.00 13.04 0.32 Asn AAT 0.00 0.000.00 Asn AAC 12.00 26.09 1.00 Met ATG 9.00 19.57 1.00 Ile ATA 1.00 2.170.08 Ile ATT 2.00 4.35 0.15 Ile ATC 10.00 21.74 0.77 Thr ACG 5.00 10.870.28 Thr ACA 2.00 4.35 0.11 Thr ACT 2.00 4.35 0.11 Thr ACC 9.00 19.570.50 Trp TGG 8.00 17.39 1.00 End TGA 1.00 2.17 1.00 Cys TGT 1.00 2.170.12 Cys TGC 7.00 15.22 0.88 End TAG 0.00 0.00 0.00 End TAA 0.00 0.000.00 Tyr TAT 1.00 2.17 0.12 Tyr TAC 7.00 15.22 0.88 Leu TTG 3.00 6.520.08 Leu TTA 1.00 2.17 0.03 Phe TTT 6.00 13.04 0.25 Phe TTC 18.00 39.130.75 Ser TCG 11.00 23.91 0.26 Ser TCA 4.00 8.70 0.09 Ser TCT 6.00 13.040.14 Ser TCC 9.00 19.57 0.21 Arg CGG 4.00 8.70 0.11 Arg CGA 4.00 8.700.11 Arg CGT 0.00 0.00 0.00 Arg CGC 9.00 19.57 0.24 Gln CAG 11.00 23.910.73 Gln CAA 4.00 8.70 0.27 His CAT 0.00 0.00 0.00 His CAC 6.00 13.041.00 Leu CTG 9.00 19.57 0.24 Leu CTA 4.00 8.70 0.11 Leu CTT 4.00 8.700.11 Leu CTC 17.00 36.96 0.45 Pro CCG 8.00 17.39 0.29 Pro CCA 7.00 15.220.25 Pro CCT 5.00 10.87 0.18 Pro CCC 8.00 17.39 0.29

Other modifications can also be made to the nucleotide sequence ofinterest to enhance its expression in duckweed. These modificationsinclude, but are not limited to, elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well characterized sequenceswhich may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence may be modified to avoid predicted hairpinsecondary mRNA structures.

There are known differences between the optimal translation initiationcontext nucleotide sequences for translation initiation codons inanimals and plants and the composition of these translation initiationcontext nucleotide sequence can influence the efficiency of translationinitiation. See, for example, Lukaszewicz et al. (2000) Plant Science154:89-98; and Joshi et al. (1997); Plant Mol. Biol. 35:993-1001. In thepresent invention, the translation initiation context nucleotidesequence for the translation initiation codon of the nucleotide sequenceof interest may be modified to enhance expression in duckweed. In oneembodiment, the nucleotide sequence is modified such that the threenucleotides directly upstream of the translation initiation codon of thenucleotide sequence of interest are “ACC.” In a second embodiment, thesenucleotides are “ACA.”

Expression of a transgene in duckweed can also be enhanced by the use of5′ leader sequences. Such leader sequences can act to enhancetranslation. One or more leader sequences may be used in combination toenhance expression of the target nucleotide sequence. Translationleaders are known in the art and include, but are not limited to,picornavirus leaders, e.g., EMCV leader (Encephalomyocarditis 5′noncoding region; Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA86:6126); potyvirus leaders, e.g., TEV leader (Tobacco Etch Virus;Allison et al. (1986) Virology 154:9); human immunoglobulin heavy-chainbinding protein (BiP; Macajak and Sarnow (1991) Nature 353:90);untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4; Jobling and Gehrke (1987) Nature 325:622); tobacco mosaicvirus leader (TMV; Gallie (1989) Molecular Biology of RNA, 23:56);potato etch virus leader (Tomashevskaya et al. (1993) J. Gen. Virol.74:2717-2724); Fed-1 5′ untranslated region (Dickey (1992) EMBO J.11:2311-2317); RbcS 5′ untranslated region (Silverthorne et al. (1990)J. Plant. Mol. Biol. 15:49-58); and maize chlorotic mottle virus leader(MCMV; Lommel et al. (1991) Virology 81:382). See also, Della-Cioppa etal. (1987) Plant Physiology 84:965. Leader sequence comprising plantintron sequence, including intron sequence from the maize dehydrogenase1 gene, the castor bean catalase gene, or the Arabidopsis tryptophanpathway gene PAT1 has also been shown to increase translationalefficient in plants (Callis et al. (1987) Genes Dev. 1:1183-1200;Mascarenhas et al. (1990) Plant Mol. Biol. 15:913-920). In oneembodiment of the present invention, nucleotide sequence correspondingto nucleotides 1222-1775 of the maize alcohol dehydrogenase 1 gene(GenBank Accession Number X04049), set forth in SEQ ID NO: 1, isinserted upstream of the nucleotide sequence of interest to enhance theefficiency of its translation. In another embodiment, the expressionvector contains the leader from the Lemna gibba ribulose-bis-phosphatecarboxylase small subunit 5B gene (Buzby et al. (1990) Plant Cell2:805-814).

It is recognized that any of the duckweed expression-enhancingnucleotide sequence modifications described above can be used in thepresent invention, including any single modification or any possiblecombination of modifications. The phrase “modified for enhancedexpression in duckweed” as used herein refers to a nucleotide sequencethat contains any one or any combination of these modifications.

D. Signal Peptides

Secreted proteins are usually translated from precursor polypeptidesthat include a “signal peptide” that interacts with a receptor proteinon the membrane of the endoplasmic reticulum (ER) to direct thetranslocation of the growing polypeptide chain across the membrane andinto the endoplasmic reticulum for secretion from the cell. This signalpeptide is often cleaved from the precursor polypeptide to produce a“mature” polypeptide lacking the signal peptide. In an embodiment of thepresent invention, a biologically active polypeptide is expressed induckweed from a nucleotide sequence that is operably linked with anucleotide sequence encoding a signal peptide that directs secretion ofthe polypeptide into the culture medium. Plant signal peptides thattarget protein translocation to the endoplasmic reticulum (for secretionoutside of the cell) are known in the art. See, for example, U.S. Pat.No. 6,020,169 to Lee et al. In the present invention, any plant signalpeptide can be used to in target polypeptide expression to the ER. Insome embodiments, the signal peptide is the Arabidopsis thaliana basicendochitinase signal peptide, the extensin signal peptide (Stiefel etal. (1990) Plant Cell 2:785-793), or the rice α-amylase signal peptide(SEQ ID NO:8; amino acids 1-31 of NCBI Protein Accession No. AAA33885).In another embodiment, the signal peptide corresponds to the signalpeptide of a secreted duckweed protein.

Alternatively, a mammalian signal peptide can be used to targetrecombinant polypeptides expressed in genetically engineered duckweedfor secretion. It has been demonstrated that plant cells recognizemammalian signal peptides that target the endoplasmic reticulum, andthat these signal peptides can direct the secretion of polypeptides notonly through the plasma membrane but also through the plant cell wall.See U.S. Pat. Nos. 5,202,422 and 5,639,947 to Hiatt et al.

In some embodiment, the nucleotide sequence encoding the signal peptideis modified for enhanced expression in duckweed, utilizing anymodification or combination of modifications disclosed in section Babove for the nucleotide sequence of interest. For example, a duckweedoptimized sequence encoding the signal peptide from rice α-amylase isshown in SEQ ID NO:7. This sequence contains approximately 93% duckweedpreferred codons.

The secreted polypeptide can be harvested from the culture medium by anyconventional means known in the art and purified by chromatography,electrophoresis, dialysis, solvent-solvent extraction, and the like.

E. Transformed Duckweed Plants and Duckweed Nodule Cultures

The stably transformed duckweed utilized in this invention can beobtained by any method known in the art. In one embodiment, the stablytransformed duckweed is obtained by one of the gene transfer methodsdisclosed in U.S. Pat. No. 6,040,498 or U.S. Patent Publication Numbers20030115640, 20030033630, or 20020088027; each of which is hereinincorporated by reference. These methods include gene transfer byballistic bombardment with microprojectiles coated with a nucleic acidcomprising the nucleotide sequence of interest, gene transfer byelectroporation, and gene transfer mediated by Agrobacterium comprisinga vector comprising the nucleotide sequence of interest. In someembodiments, the stably transformed duckweed is obtained via any one ofthe Agrobacterium-mediated methods disclosed in U.S. Pat. No. 6,040,498to Stomp et al. The Agrobacterium used is Agrobacterium tumefaciens orAgrobacterium rhizogenes.

Stably transformed duckweed plants may also be obtained by chloroplasttransformation. See, for example, U.S. provisional patent applicationNo. 60/492,179, filed Aug. 1, 2003, entitled “Chloroplast transformationof duckweed.” Stably transformed duckweed lines may also be producedusing plant virus expression vectors. See, for example, U.S. Pat. No.6,632,980 and Koprowski and Yusibov (2001) Vaccine 19:2735-2741.

It is preferred that the stably transformed duckweed plants utilized inthese methods exhibit normal morphology and are fertile by sexualreproduction. Preferably, transformed plants of the present inventioncontain a single copy of the transferred nucleic acid, and thetransferred nucleic acid has no notable rearrangements therein. Alsopreferred are duckweed plants in which the transferred nucleic acid ispresent in low copy numbers (i.e., no more than twelve copies, no morethan eight copies, no more than five copies, alternately, no more thanthree copies, as a further alternative, fewer than three copies of thenucleic acid per transformed cell).

EXPERIMENTAL

The following examples are offered for purposes of illustration, not byway of limitation.

Expression Constructs for the Production of Plasminogen andMicroplasminogen in Duckweed

The expression vector used in the present examples is a modified versionof pBMSP-1, which is described in U.S. Pat. No. 5,955,646, hereinincorporated by reference. The transcriptional cassette of the vectorcontained three copies of a transcriptional activating nucleotidesequence derived from the Agrobacterium tumefaciens octopine synthaseand, an additional transcriptional activating nucleotide sequencederived from the Agrobacterium tumefaciens mannopine synthase gene, apromoter region derived from the Agrobacterium tumefaciens mannopinesynthase gene, a polylinker site for insertion of the nucleotidesequence encoding the polypeptide of interest, and a terminationsequence derived from the Agrobacterium tumefaciens nopaline synthasegene (see, van Engelen et al. (1995) 4:288-290; Ni et al. (1995) PlantJ. 7:661-76; and Luehrsen et al. (1991) Mol. Gen. Genet. 225:81-93, eachof which is herein incorporated by reference).

The expression vector also contained a nucleotide sequence coding forgentamycin acetyltransferase-3-I, aacC1, Wohlleben et al. (1989) Mol.Gen. Genet. 217:202-208) encoding gentamycin resistance as a selectablemarker. Transcription of the selectable marker sequence is driven by apromoter derived from the Agrobacterium tumefaciens nopaline synthase IIgene.

The expression vector additionally contains the leader from theribulose-bis-phosphate carboxylase small subunit 5B gene of Lemna gibba(nucleotides 689-751 of NCBI Accession No. S45167, Buzby et al. (1990)Plant Cell 2:805-814) and a nucleotide sequence corresponding tonucleotides 1222-1775 of the maize alcohol dehydrogenase gene (GenBankAccession Number X04049) inserted between the promoter and thepolylinker. This intron sequence is shown in SEQ ID NO:1, and the leadersequence is shown in SEQ ID NO:2.

Transformation of Duckweed

Duckweed fronds or duckweed nodule cultures (derived from Lemna minorstrain 8627 in these examples) were transformed with the expressionconstructs described above using Agrobacterium-mediated transformationmethods. Agrobacterium tumefaciens strain C58Z707, a disarmed, broadhost range C₅₋₈ strain (Hepburn et al. (1985) J. Gen. Microbiol.131:2961-2969) is used for transformation in these examples. Theexpression constructs described above were mobilized into A. tumefaciensby electroporation, or by a triparental mating procedure using E. colimM294 harboring the mobilizing plasmid pRK2013 (Hoekema et al. (1983)Nature 303: 179-180; Ditta et al. (1980) Proc. Natl. Acad. Sci. USA 77:7347-7350). C58Z707 strains comprising the expression constructsdescribed above are streaked on AB minimal medium (Chilton et al.,(1974) Proc. Nat. Acad. Sci. USA 71: 3672-3676) or in YEB or LB medium(1 g/L yeast extract, 5 g/L beef extract, 5 g/L peptone, 5 g/L sucrose,0.5 g/L MgSO₄) containing streptomycin at 500 mg/L, spectinomycin at 50mg/L and kanamycin sulfate at 50 mg/L and grown overnight at 28° C.

Duckweed nodule cultures for transformation were produced as follows.Duckweed fronds were separated, the roots are cut off with a sterilescalpel, and the fronds are placed, ventral side down, on Murashige andSkoog medium (catalog number M-5519; Sigma Chemical Corporation, St.Louis, Mo.) pH 5.6, supplemented with 5 μM 2,4-dichlorophenoxyaceticacid, 0.5 μM 1-Phenyl-3(1,2,3-thiadiazol-5-yl) urea thidiazuron (SigmaP6186), 3% sucrose, 0.4 Difco Bacto-agar (Fisher Scientific), and 0.15%Gelrite (Sigma). Fronds were grown for 5-6 weeks. At this time, thenodules (small, yellowish cell masses) appeared, generally from thecentral part of the ventral side. This nodule tissue was detached fromthe mother frond and cultured in Murashige and Skoog medium supplementedwith 3% sucrose, 0.4% Difco Bacto-agar, 0.15% Gelrite, 1 μM2,4-dichlorophenoxyacetic acid, and 2 μM benzyladenine.

Duckweed nodule cultures were transformed as follows. The appropriateAgrobacterium tumefaciens strain was grown on potato dextrose agar orYEB or LB agar with 50 mg/L kanamycin and 100 μM acetosyringone, andresuspended in Murashige and Skoog medium supplemented with 0.6 MMannitol and 100 μM acetosyringone. Nodule culture tissue was inoculatedby immersing in the solution of resuspended bacteria for 1-2 minutes,blotted to remove excess fluid, and plated on co-cultivation mediumconsisting of Murashige and Skoog medium supplemented with auxin andcytokinin optimized to promote nodule growth and 100 μM acetosyringone.See, Yamamoto et al. (2001) In Vitro Cell Dev. Biol. Plant 37:349-353.

For selection, nodule culture tissue was transferred to regenerationmedium; 0.5 X Schenk and Hildebrandt medium supplemented with 1% sucrose0.4% Difco Bacto-Agar, 0.15% Gelrite 500 mg/L cefotaxime, and 6 mg/Lgeneticin and cultured for approximately 6-8 weeks under continuouslight (20-40 μM/m²·sec). The nodule tissue was transferred every 7 daysto fresh culture medium. Selection is complete when the nodule tissueshows vigorous growth on the selection agent.

The following examples demonstrate the expression of biologically activeplasminogen and microplasminogen in duckweed.

Example 1 Production of Plasminogen

Human plasminogen was expressed in duckweed as follows. A syntheticduckweed-codon optimized sequence encoding human plasminogen wasinserted into the expression vector described above. The amino acidsequence of the encoded plasminogen is given in SEQ ID NO:4, and theduckweed-optimized coding sequence is given in SEQ ID NO:3. Theexpression vector also contained a duckweed optimized coding sequenceencoding the rice alpha amylase signal peptide inserted 5′ of theplasminogen coding sequence. The nucleotide sequence encoding the signalpeptide was operably linked to the nucleotide sequence encoding humanplasminogen such that the two coding sequences would be translated asone protein. The duckweed optimized coding sequence encoding the ricealpha amylase signal peptide is given in SEQ ID NO:7, and the encodedpeptide is given in SEQ ID NO:8. This plasminogen expression vector isreferred to as BAP01 in this example.

Duckweed lines transformed with the BAP01 expression vector weregenerated as described above. Due to the large size of plasminogen, theprimary screening of transgenic lines included analysis of both mediaand tissue homogenates. The vast majority of expressed protein wasretained in the tissue. A total of 81 lines were generated andplasminogen accounted for as much as 6% of the total soluble protein inplant extracts (as measured by ELISA). FIG. 1 represents the level ofplasminogen measured in 56 of the transgenic lines. These levels wereobtained from plants grown for 2 weeks in research vessels. Commerciallyavailable plasminogen was used as the standard in this assay.

The activity of the duckweed-expressed plasminogen was determined fortissue extracts from 7 different independent transgenic lines. Theduckweed-expressed plasminogen was activated by streptokinase to producean active complex. Activation by streptokinase does not involve theformation of plasmin, but occurs by a conformational shift that resultsfrom formation of a streptokinase/plasminogen complex. The activity ofthe resulting complex was then determined by assaying for cleavage ofthe chromogenic substrate Glu-Phe-Lys-pNA (available from ChromgenixInstrumentation Laboratory SpA, Milano Italy) shown in SEQ ID NO:9(pNA=p-Nitroanilide) at 405 nm. (Gram J. and Jespersen J. Thromb.Haemost. 53, 255-259 (1985) and Robbins, K. C. Semin. Thromb. Haemost.13 (2), 131-138 (1987)). For each of the seven transgenic lines tested,the activity level was closely correlated with the protein expressionlevels as determined by ELISA, indicating that the plasminogen producedin Lemna had a similar specific activity to that of the commerciallyavailable control protein.

It was noted that many of the duckweed lines that were engineered toover express plasminogen underwent rapid senescence. However, it wasdetermined that senescence in these lines could be reduced by increasingthe aeration and media volume during plant culture. In addition,altering the inoculum density can also influence the plant health. Theculture of eight transgenic duckweed lines was scaled-up, and one ofthese lines, BAP01-B1-95, was selected for further analysis. This linewas selected based on biomass accumulation, plasminogen expression level(3.3% of total soluble protein levels in crude plant extracts asdetermined by ELISA), and overall plant health.

Plasminogen was harvested from the BAP01-B1-95 line as follows. Bulkduckweed tissue was homogenized, clarified by centrifugation, filteredthrough a 0.22 μM filter, passed through a Dowex ion exchange resincolumn (available from Dow Chemical, Midland, Mich.), and then affinitypurified by lysine Sepharose chromatography. Bound material was elutedfrom the affinity column with 8-amino caproic acid. Crude tissueextracts obtained from these plants contained plasminogen as 3.3% of thetotal soluble protein as measured by ELISA.

The activity of the duckweed-produced plasminogen following activationby tPA urokinase, and streptokinase was also determined. Activity ofplasminogen activated with either tPA or urokinase produced activatedplasmin as shown in FIG. 2. Western blotting analysis showed that boththe heavy and light chain of the plasmin produced by activation theduckweed-produced plasminogen co-migrated with the commerciallyavailable plasmin used as a control (FIG. 6). Activation withstreptokinase also produced activated plasminogen as shown in SEQ IDNO:3. In this assay, plasminogen is activated by streptokinase toproduce an active complex, which then cleaves a chromogenic substrate(Coamatic® brand plasminogen kit, DiaPharma, West Chester, Ohio). FIG. 3shows that for the lines tested, quantitation by ELISA and streptokinaseactivity assay gave comparable values, indicating that Lemna-producedplasminogen has a similar specific activity to the control protein.

The size of the duckweed-produced plasminogen was determined by Westernblotting using an anti-plasminogen antibody available from AmericanDiagnostica, Inc. Greenwich, Conn. This analysis showed that 60% of theplasminogen produced in duckweed was full length N-terminal sequencingof the duckweed produced plasminogen showed further processing producinga polypeptide missing the first 74 amino acids. In human serum,plasminogen is isolated as a mixture of unprocessed ‘glu-plasminogen’and several processed versions defined as ‘lys-plasminogen’ where 69,77, or 78 N-terminal amino acids are removed. Such a cleavage has noeffect on activity.

Because references in the prior art have reported difficulty inproducing stable plasminogen in recombinant systems, the stability ofplasminogen produced in the Lemna system was tested. FIG. 4 demonstratesthe stability of human plasminogen added to a Lemna tissue extract. Thefigure demonstrates that the human plasminogen retained almost all ofits activity even after an overnight incubation in the Lemna extract.FIG. 5 demonstrates the stability of Lemna-produced plasminogen in Lemnatissue extracts following a freeze-thaw cycle. The figure demonstratesthat the Lemna-produced protein is stable following a freeze-thaw cycle.

Example 2 Production of Microplasminogen in Duckweed

Human microplasminogen was expressed in duckweed as follows. A syntheticduckweed-codon optimized sequence encoding human microplasminogen wasinserted into the expression vector described above. The amino acidsequence of the encoded microplasminogen is given in SEQ ID NO:6, andthe duckweed-optimized coding sequence is given in SEQ ID NO:5. Theexpression vector also contained a duckweed optimized coding sequenceencoding the rice alpha amylase signal peptide inserted 5′ of theplasminogen coding sequence. The nucleotide sequence encoding the signalpeptide was operably linked to the nucleotide sequence encoding humanmicroplasminogen such that the two coding sequences would be translatedas one protein. The duckweed optimized coding sequence encoding the ricealpha amylase signal peptide is given in SEQ ID NO:7, and the encodedpeptide is given in SEQ ID NO:8. This microplasminogen expression vectoris referred to as BAMP01 in this example.

Duckweed lines transformed with the BAMP01 expression vector weregenerated as described above. The majority of the expressedmicroplasminogen was secreted into the culture medium. 79 transgeniclines were screened to determine microplasminogen expression levels.FIG. 7 shows the levels of microplasminogen expressed in these lines.The first set of 42 lines were grown for 7 days, while the second set of37 lines were grown for six days. It should be noted that duckweed isunique in that it grows in very dilute aqueous media inorganic mediawith a very low protein content, distinguishing it from typefermentation based systems and mammalian cell-based expression systems.The duckweed growth media typically contains only 30 mg/L of host plantproteins. This low level of host plant protein provides advantages forthe downstream purification of the secreted protein.

One of the BAMP01 transformed duckweed lines, BAMP01-B1-58, was selectedfor further study. Microplasminogen was harvested from the culture mediaof the BAMP01-B1-58 line as follows. The culture media was process byultrafiltration and diafiltration and then passed over a Dowex ionexchange resin column (available from Dow Chemical, Midland, Mich.) toremove low molecular weight plant metabolites. The concentration beforeconcentrating of microplasminogen in the crude aqueous media wasapproximately 20 mg/L as determined by quantitative Western blotting.

The size of the duckweed-produced microplasminogen was determined byWestern blotting using an anti plasminogen antibody available from(American Diagnostica Inc. Greenwich, Conn.). This analysis showed thatmost of the microplasminogen produced in duckweed was full length. Anintact N-terminus was confirmed also by N-terminal sequencing

The activity of the duckweed-expressed microplasminogen was determinedfor the BAMP01-B1-58 transgenic line following activation bystreptokinase to produce an active complex as described above forplasminogen. The duckweed-produced microplasminogen showed some level ofactivity in the absence of streptokinase, and a significant increase inactivity following activation by streptokinase.

Like plasminogen, microplasminogen can be activated by urokinase andtPA; however, only the B-chain is produced. Western blot analysisconfirmed the production of the B chain of plasmin following activationof duckweed-produced microplasminogen with either urokinase or tPA.

To confirm the activity of plasmin from Lemna-produced microplasminogen,a gelatin zymogram was run on concentrated media following activation bytPA. FIG. 8 shows the presence of an active proteolytic band that is notpresent in concentrated control media.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended embodiments.

1. A stably transformed duckweed plant, duckweed plant cell, or duckweednodule, wherein said duckweed plant or duckweed plant cell or nodule isstably transformed with a nucleic acid molecule comprising a nucleotidesequence encoding plasminogen, wherein said nucleic acid moleculecomprises: a) duckweed-preferred codons in the coding sequence for saidplasminogen; b) an operably linked nucleotide sequence comprising aplant intron that is inserted upstream of the coding sequence for saidplasminogen; c) an operably linked nucleotide sequence coding for asignal peptide, said signal peptide-encoding sequence inserted betweensaid intron and said plasminogen-encoding sequence; and d) an operablylinked nucleotide sequence comprising the translation leader sequencefrom the ribulose-bis-phosphate carbosylase small subunit 5B gene ofLemna gibba.
 2. The stably transformed duckweed plant or duckweed plantcell or nodule according to claim 1, wherein said signalpeptide-encoding sequence is from a rice alpha-amylase gene.
 3. Thestably transformed duckweed plant or duckweed plant cell or noduleaccording to claim 2, wherein said signal peptide-encoding sequence isSEQ ID NO:8.
 4. The stably transformed duckweed plant or duckweed plantcell or nodule according to claim 1, wherein the nucleotide sequenceencoding plasminogen comprises between 70-100% duckweed-preferredcodons.
 5. The stably transformed duckweed plant or duckweed plant cellor nodule according to claim 1, wherein said plant intron is an intronfrom maize alcohol dehydrogenase 1 gene.
 6. The stably transformedduckweed plant or duckweed plant cell or nodule according to claim 5,wherein said plant intron is SEQ ID NO:1.
 7. The stably transformedduckweed plant or duckweed plant cell or nodule according to claim 1,wherein said translation leader sequence is SEQ ID NO:2.
 8. The stablytransformed duckweed plant or duckweed plant cell or nodule according toclaim 1, wherein said plasminogen is human plasminogen.
 9. The stablytransformed duckweed plant or duckweed plant cell or nodule according toclaim 8, wherein said plasminogen has at least 95% sequence identitywith SEQ ID NO:4.
 10. The stably transformed duckweed plant or duckweedplant cell or nodule according to claim 1, wherein at least 2% ofsoluble protein in the duckweed plant or duckweed plant cell or noduleis stable plasminogen.
 11. The stably transformed duckweed plant orduckweed plant cell or nodule according to claim 10, wherein at least 3%of soluble protein in the duckweed plant or duckweed plant cell ornodule is stable plasminogen.
 12. The stably transformed duckweed plantor duckweed plant cell or nodule according to claim 11, wherein at least4% of soluble protein in the duckweed plant or duckweed plant cell ornodule is stable plasminogen.
 13. The stably transformed duckweed plantor duckweed plant cell or nodule according to claim 1, wherein saidduckweed plant or duckweed plant cell or nodule is from a genus selectedfrom the group consisting of the genus Spirodela, genus Wolffia, genusWolfiella, genus Landoltia and genus Lemna.
 14. The stably transformedduckweed plant or duckweed plant cell or nodule according to claim 1,wherein said duckweed plant or duckweed plant cell or nodule is from aspecies selected from the group consisting of Lemna minor, Lemnaminiscula, Lemna aequinoctialis, and Lemna gibba.
 15. A stablytransformed duckweed plant, duckweed plant cell, or duckweed nodule,wherein the duckweed plant or duckweed plant cell or nodule is stablytransformed with a nucleic acid molecule comprising a nucleotidesequence encoding plasminogen, wherein the nucleotide sequence is SEQ IDNO:3.
 16. The stably transformed duckweed plant or duckweed plant cellor nodule according to claim 15, wherein said nucleic acid moleculecomprises at least one attribute selected from the group consisting of:(a) an operably linked nucleotide sequence comprising a plant intronthat is inserted upstream of the nucleotide sequence encodingplasminogen; and (b) an operably linked nucleotide sequence comprising atranslation leader sequence.
 17. A stably transformed duckweed plant,duckweed plant cell, or duckweed nodule, wherein said duckweed plant orduckweed plant cell or nodule is stably transformed with a nucleic acidmolecule comprising a nucleotide sequence encoding plasminogen, saidplasminogen having at least 95% sequence identity with SEQ ID NO:4,wherein said nucleic acid molecule comprises: a) duckweed-preferredcodons in the coding sequence for said plasminogen; b) an operablylinked nucleotide sequence comprising a plant intron that is insertedupstream of the coding sequence for said plasminogen, wherein said plantintron is SEQ ID NO:1; and c) an operably linked nucleotide sequencecomprising a translation leader sequence, wherein said translationleader sequence is SEQ ID NO:2.
 18. A composition comprisingduckweed-produced plasminogen.